The immobilization of entities (such as enzymes, antibodies, proteins, DNA, nucleotides, PNA, carbohydrates, fatty acids, lectins, peptides, receptors, chromophores, fluorophores, chemiluminescent compounds, dendrimers, J or H aggregates, cells, bacteria, viruses, whole prokaryotic or eukaryotic organisms, membranes (synthetic or natural), fullerenes, nanotubes and the like) can be achieved by simple covalent reaction with an activated solid surface. For example, particles (e.g., micro- and nano-spheres; metal particles comprised of one or more metals with any size, shape, or composition; —semiconductor particles, molecularly imprinted polymers (MIPS), magnetic particles; or dyed materials) or microtiter plates are a common solid matrix in many immobilization systems. Preparing and maintaining the active, functionalized surface is an important factor to assure immobilization of sufficient biological material for development of a sensitive assay. Current immobilization procedures of biomolecules on solid surfaces generally involve reactions of an activated amino or carboxyl group derivatized solid surface with amino- or thiol-modified biomolecules. After activation, or after introduction of a functionalized spacer, these groups offer direct attachment sites. Most of these functional groups (such as NHS esters, isothiocyanates, etc.) are prone to hydrolysis in an aqueous environment and become non-reactive (i.e., chemically inactive) in a matter of less than an hour.
Reactive or functionalized microspheres are conventionally produced via copolymerization of suitably functionalized monomers, or via post-chemical modification of preformed microspheres. Post-functionalization is a popular method for preparing reactive particles as earlier described by Upson (J. Polym. Sci., Polym. Symp. 1985, 72, 45).
In the last three decades the advancements in the field of affinity chromatography, solid-phase synthesis, and immobilization of bio-macromolecules, such as proteins, oligonucleotides and the like, have led to microsphere-based biomedical applications. More recent work on the production and evaluation of a variety of tailor-made particles has been reported by several groups including Margel et al., (J. Polym. Sci. 1991, A-29, 347-355; Anal. Biochem. 1981, 128, 342-350), Ugelstad et al., (Makromol. Chem. 1979, 180, 737-44; Adv. Colloid Interface Sci. 1980, 13, 102-140), and Rembaum et al. (Br. Polym. J. 1978, 10, 275-280; J. Macromol. Sci. Chem. 1979, A-13, 603-632). A review by R. Arshady, (Biomaterials, 1993, 14, 5-15) describes the synthesis and physico-chemical properties of reactive and labeled microspheres.
Assays based on fluorescent microspheres for multiplexed analysis have been reported by several groups (Fulton et al., Clin. Chem. 1997, 43, 1749-56; Kettman et al., Cytometry, 1998, 33, 234-43; McDade et al., Med. Dev. Diag. Indust. 1997, 19(4), 75-82; McHugh, Methods Cell Biol. 1994, 42, 575-95; Nikiforov et al., Nucleic Acid Res. 11994, 22, 4167-75; U.S. Pat. No. 6,449,562; U.S. Pat. No. 5,981,180; U.S. Pat. No. 6,046,807; U.S. Pat. No. 6,057,107; U.S. Pat. No. 6,268,222; U.S. Pat. No. 6,366,354; U.S. Pat. No. 6,411,904; U.S. Pat. No. 5,736,330; U.S. Pat. No. 6,139,800).
Fray et al have reported a strategy in which particles are pre-activated with hydrolysis-resistant aldehyde functional groups but low reaction yields of <8% have been observed with these microspheres (Bioconjugate Chem. 1999, 10, 562-71). Milton of Beckman Coulter, Inc. has reported a reaction between an acyl fluoride activated polymer-surface and an amino derivatized biomolecule at room temperature (U.S. Pat. No. 6,146,833; Nov. 14, 2000). The use of fluorophenyl resins in the solid phase synthesis of amides, peptides, hydroxamic acids, amines, urethanes, carbonates, sulfonamides and alpha-substituted carbonyl compounds has been published (WO 99/67228).
Medvedkin et al. have used sulfo-tetrafluorophenyl activated esters in peptide synthesis and demonstrated their reactivity combined with good stability under aqueous storage conditions (Bioorg. Khim. 1995, 21(9), 684-90). Apparently, the pre-activation of polystyrene surfaces with this reagent has not yet been reported prior to the present application.
Hoechst claimed the use of reactive vinyl sulfone (VS)-modified dyes for dyeing of cellulose and wool fibers in 1950 (DBP 960,534). A review by Siegel gives a complete account of reactive dyes based on vinyl sulfones (VS) and its protected 2-sulfatoethyl and 2-thiosulfatoethyl sulfones (E Siegel in The Chemistry of Synthetic Dyes Vol. VI, (Ed K Venkataraman); 2-108, Academic Press, 1972). Sterling Winthrop Inc, has demonstrated modification of proteins with PEG-supported vinyl sulfone (U.S. Pat. No. 5,414,135).
The most frequently used method to immobilize biomolecules (such as oligonucleotides, proteins, or carbohydrates) onto fluorescent microspheres is by activating surface carboxy groups. The activation requires excess EDC and a coupling pH of 4 to 6 The reaction involves the intermediate formation of an activated O-acylurea derivative between the carbodiimide and carboxyl functions. A subsequent nucleophilic attack by the primary nitrogen of the amino-groups of the biomolecule brings about the formation of the amide linkage with the release of the substituted urea. The optimum pH for the formation of O-acylurea is about pH 4-5. The intermediate has an extremely short half-life and rapidly undergoes hydrolysis or rearranges to give the N-acylurea adduct. The primary amino group of the nucleophile is predominantly protonated at about pH 4-5 and is thus mostly unreactive. These limitations can severely restrict coupling yields. At low pH, the nucleic acid bases undergo intensive protonation. Such type of protonation induces a DNA melting that exposes the hydrophobic core of the helix, enhancing nonspecific hydrophobic interactions with the solid matrix. Despite these drawbacks, EDC-mediated coupling currently is the major mode of covalent immobilization of biomolecules to solid surfaces. (Hermanson, G. T. in Bioconjugate Techniques, Academic Press; N.Y. 1996; Andreas Frey et. al., Bioconjugate Chem. 1999, 10, 562-71; Maxime A. Gilles et. al., Anal. Biochem., 1990, 184, 244-48; Vivien W. F. Chan et. al., Biochem. Biophys. Res. Communications., 1988, 151(2), 709-16; Ivan L. Valuev et al., Biomaterials, 1998, 19, 41-43.)
The citations of the various references described above and throughout this application are not to be taken as admissions that these references constitute prior art for the present invention. However, each of the cited references is incorporated in its entirety by reference in the present application.